1. Field of the Invention
The invention relates to a method and associated apparatus for performing solid phase assays having special utility for use in conjunction with automatic pipetting equipment.
2. Description of the Prior Art
In recent years, numerous techniques have been employed in the area of laboratory diagnostics to simplify operating procedures of existing methods and to provide new methods of improved speed, sensitivity and accuracy. In particular, solid phase reactions have been especially valuable in simplifying the manipulations of prior art procedures and making possible procedures that could not be performed with conventional homogeneous phase reactions. However, solid phase assay techniques heretofore have suffered from the disadvantage of requiring hand manipulation of small volumes which inherently are subject to a wide range of laboratory error.
A solid phase reaction is generally carried out between one reactant, the fixed component, immobilized on the surface of an insoluble support, and a second reactant, the mobile component, in solution. The reaction occurs when a molecule or molecular arrangement of the mobile reactant, in the course of diffusion, collides with a molecule of the fixed reactant immobilized on the surface of the solid support matrix. The reaction may be a conventional chemical reaction, a binding of the mobile component by the fixed component as in an immunochemical reaction between an antigen and an antibody, or it may be a binding of the mobile component by the fixed component accompanied by chemical transformation of one of the components such as occurs in an enzyme-catalyzed reaction. Quantitative results are obtained by measuring the formation of products or disappearance of reactants as in the case of conventional and enzyme-catalyzed reactions, and in measuring the amount of the mobile component bound or the amount of fixed component unbound, in the case of an immunochemical reaction.
Any conventional chemical reaction or enzyme-catalyzed reaction resulting in a directly or indirectly measurable change can, in principle, be carried out by solid phase techniques. Direct measurable changes include changes of pH, light absorbance in the visible and ultraviolet regions or changes in fluorescence intensity. Indirect measurements can be made whenever the primary reactants or products are not readily measurable themselves by interposing the action of a reagent to carry out further reaction steps resulting in a measurable change and by the introduction of specific separation techniques. Such strategies may be employed alone or in combinations, as is understood in the art.
Where the reaction consists solely of binding, in the absence of chemical change, techniques developed in the field of immunochemistry may be used to measure the extent of the reaction. Solid phase reactions are especially suited for immunochemical assays because the reactants in bound form may readily be removed from the solution by virtue of their attachment to the solid phase. Frequently, however, the components bound in an immunochemical reaction cannot be directly measured because they are indistinguishable by chemical methods from other substances commonly present in the same reaction mixture so that the mere disappearance of a reactive component from solution or its accumulation on the solid phase cannot be measured directly. Therefore, additional steps are taken in order to make a measurable change related to the amount of binding.
The variety of approaches taken by workers in the prior art can be grouped into two general categories. In the first of these, termed competitive or indirect immunoassays, the immobilized component is present in controlled amount and the mobile component present in unknown amount. To the unknown amount of mobile component is added a known amount of the same component which has been tagged by the addition of a measurable substituent which does not interfere with its immunochemical reactive properties. The tag may consist of a radioisotope, a chromophore, a fluorophor or an enzyme. The amount of tagged material bound immunochemically to the solid phase will depend upon the amount of untagged component in solution competing for the same binding sites. The more of the unknown present, the less will be the amount of tagged component bound.
In the second general method, termed the sandwich method or direct method, the solid phase containing an amount of immunochemically bound mobile component resulting from the first immunochemical reaction is subjected to the action of a reagent which can also bind immunochemically to the solid phase, but only at sites already occupied by the immunochemically bound mobile component. The reagent may be tagged, for example, as in the first method with a radioisotope, a fluorophor, a chromophore or an enzyme. The amount of tagged reagent bound is a direct measure of the amount of mobile component bound, which in turn is a measure of the amount of mobile component initially present in the reaction mixture. Where the tag is a radioisotope, the technique, whether competitive or noncompetitive, is termed a radioimmunoassay. When the tag is an enzyme, the assay is termed an enzyme immunoassay.
Other kinds of solid phase reactions of the type generally described hereinabove are presented by way of example. The immunoradiometric assay for quantitative determination of an antigen is conducted by first reacting a known excess of labeled antibody with the unknown amount of antigen in a homogeneous phase reaction. Subsequently, immobilized antigen in excess amount is added in order to bind the unreacted soluble labeled antibody. The amount of unknown antigen is determined by measuring the difference between the total labeled antibody and the amount bound to the solid phase. The method gives direct quantitative resuls only with univalent antigen, i.e., antigen which can only bind one molecule of antibody.
Enzyme-catalyzed reactions are conveniently carried out in solid phase systems. An enzyme immobilized on a solid phase matrix may be used to quantitatively assay for, or qualitatively detect the presence of, the substrate for the enzyme in a sample of biological material. For example, lactic acid in serum may be measured using a matrix coated with lactic dehydrogenase. Similarly, urea may be assayed using a solid phase insert bearing immobilized urease. In additional to clinical applications, enzyme assays may be used for quality control monitoring of industrial process steps and also for carrying out process steps. As an example of the former, immobilized penicillinase could be employed in an assay to monitor the quality of penicillin produced during the process of manufacturing the drug. As an example of the latter, immobilized proteases or nucleuses could be useful to remove or inactivate contaminating proteins or nucleic acids.
The presence of an enzyme of clinical significance in a sample of biological material may also be assayed by providing a substrate for the enzyme immobilized on a solid phase matrix. An example of an assay which could be adapted for use in this fashion is the method disclosed in U.S. patent application Ser. No. 795,497 of James W. Ryan and Alfred Chung. A lysozyme assay, in which radioactively labeled Micrococcus lysodeikticus is covalently bound to the surface of a solid phase matrix, further exemplifies the use of an immobilized substrate in an enzyme reaction.
Further examples of useful solid phase reactions are provided by the specific binding reactions of certain proteins. These include, for example, beta-lactoglobulin, which specifically binds folic acid, specific receptor proteins capable of binding hormones, such as the receptor substance purified from rat mammory tumor cells which specifically binds prolactin and the variety of plant proteins such as concanavalin A, which are capable of specifically binding certain carbohydrates.
Conventional chemical reactants may be designed for use in solid phase reactions. Solid phase reactants capable of forming colored complexes, as by the formation of glycosyl derivatives or by diazo coupling to a reagent immobilized on the surface of a solid phase matrix could be devised for use, either alone or in combination with an enzyme-catalyzed reaction, to provide for a color change on the surface of the matrix. Also, ion exchange reactions may conveniently be conducted using a solid phase matrix of the present invention. The foregoing examples are illustrative only and additional possibilities will be apparent to those having ordinary skill in the appropriate art.
In such solid phase technology, the reagent or reagents used in the procedure are immobilized by being coated or bonded, either covalently or by adsorption to the solid phase material, which is then wetted by the sample to be tested. The manner of coupling such reagents to the solid phase material is known. See, for example, the disclosures in U.S. Pat. No. 3,652,761, No. 3,879,262 and No. 3,896,217.
Examples of commonly used slid phase materials include, but are not limited to, glass or polymeric tubes which are coated with the reagent or reagents on their internal surfaces; polymeric coated sticks; micro and macro beads formed of polymers and of glass and porous matrices.
U.S. Pat. No. 3,826,619 discloses a solid phase matrix used for qualitative testing. The disclosed matrix differs substantially from that of the present invention. First, it may only be used to detect the presence or absence of antigens and not for measuring the quantity of antigens like the present invention. Second, the physical form of the matrix as exemplified by FIG. 5 of U.S. Pat. No. 3,826,619 is not suitable for use in conjunction with automatic pipetting equipment because there is no place for the automated insertion of a fluid dispensing device such as in the present invention. Moreover, the disclosed matrices have never been commercially used.
Coated tubes are exemplified by the Immunotube.TM. system marketed by Smith Kline Instruments of Sunnyvale, Calif.; the Rianen.TM. system of New England Nuclear, North Billerica, Mass.; the SPAC.TM. system of Mallinkrodt of St. Louis, Mo.; and the tubes described in U.S. Pat. No. 3,867,517 issued Feb. 18, 1975 to Ling.
The StiQ.TM. matrix of International Diagnostic Technology Co. of Santa Clara, Calif. is a widely used stick type solid phase matrix.
The Biological Products Division of Corning Glass Works in Medfield, Mass., markets the Immo Phase.TM. system which exemplifies the coated bead system.
Immunochemical assays are highly useful in clinical research and diagnosis. They are highly specific, owing to the highly selective nature of antigen-antibody reactions. The antigen-antibody binding is very tight so that once the binding reaction has had an opportunity to occur, the limit of detectability is determined by the measurability with which the tag can be detected. Immunochemical assays are exceedingly versatile, owing to the fact that they can be used to measure substances which lack any measurable chemically distinguishable features. Because of these desirable attributes, there has been considerable interest in improving the ease of manipulation, sensitivity and applicability of immunochemical assays. The development of solid phase immunoassays has been one of the major advances in the field.
When small volume samples are used, laboratory errors are introduced because control conditions such as pH and volume in the sample are hard to maintain and because it is difficult to insure that the solid phase is uniformly wetted during every similar assay.
As previously stated, the solid phase systems currently being used employ coated tubes, coated sticks, coated beads and sponges as solid phase configurations.
The introduction of solid phase technology has permitted the performance of novel procedures that were heretofore extremely difficult using free solution technology. An exemple of this is the sandwich assay technique described hereinabove. To be carried out in homogeneous solution, the sandwich technique would require a large excess of one of the reactants. More importantly, separation of the first antigen-antibody complex from a homogeneous phase solution requires the use of sophisticated physical-chemical techniques, especially if the antigen is relatively small compared to the antibody and molecular weight differences between free antibody and complexed antibody are slight. In contrast, the separation procedure in a solid phase system is a matter of the utmost simplicity. As will be described below, one of the primary advantages of solid phase technology, the ease of separating the solid and liquid phases, is maximized in the practice of the present invention, which provides extremely simple means for separating the phases.
While, in theory, solid phase technology offers numerous advantages over free solution or homogeneous systems, it does have certain limitations due principally to the solid phase configurations heretofore used. For example, since at least one of the reagents in a solid phase system is effectively immobilized by being bound to the surface, the reaction rate of solid phase systems is generally slower than that of homogeneous or free solutions systems. Additionally, there is normally a maximum amount of reagent which can be bound to the solid phase surface, the maximum amount being generally dependent upon the surface area, the purity of the reagent and the specific procedure used to bind the reagent to the surface. Optimally, as much as possible of the surface area of the solid material should be coated so as to increase the reaction rate and decrease the reaction time.
The earliest solid phase systems devised were test tubes coated on the inside surface. Commercial examples of coated tube technology include the Immunotube.TM. system marketed by Smith Kline Instruments of Sunnyvale, Calif., and the Rianen.TM. system of New England Nuclear, North Billerica, Mass., and the tubes described in U.S. Pat. No. 3,867,517 issued Feb. 18, 1975 to Ling. Although coated tube systems have proven useful for immunoassay purposes, they fail to exploit the full range of potential advantages offered by solid phase systems. A principal disadvantage is that the surface to volume ratio is relatively low and reaction kinetics may be further hindered by the fact that the reactive surface is located at the boundary of the solution volume, which may be relatively remote from the main body of the solution. Therefore, the average distance between mobile reactants and the reactive surface is large. In addition, each test tube must be coated separately and this constraint is likely to result in variations from tube to tube in the amount of coating material applied and ultimately in the assay results. It is also relatively expensive. Reactions conducted in coated tubes are subject to errors caused by convection in the reaction fluid. Results varying as much as 10-fold can be caused by convection in these systems.
Attempts to improve on the performance of coated tubes have led to a variety of systems designed to increase the surface to volume ratio of the solid phase system. These methods have included providing highly convoluted surfaces, reducing the volume of liquid required and providing surfaces of finely divided material.
The SPAC.TM. system of Mallinkrodt Chemical Company is basically a coated tube system which exemplifies the strategy of providing a convoluted surface to increase surface area in the coated tube format. Additionally, the tubes are provided with a detachable lower section which may be batch coated to achieve greater uniformity from tube to tube. A consequence of the batch immobilization on coated tube bottoms is that the outsides as well as the insides of the tubes become coated. This makes it difficult for the laboratory technician to work with the tubes without coming into contact with whatever material is coated on their surface and valuable immunological reactants are wasted. The convoluted surface area is said to increase by 3-4 times the amount of reactive surface available. However, the reactive surface remains at the periphery of the solution, which may be suboptimal geometry from the standpoint of the average diffusion distance from the solution to the reaction surface. Due to the complexity of the surface, difficulties in washing the surface free of contaminating substances may be encountered. As with coated tube systems in general, the SPAC.TM. system is likely to be sensitive to convection currents which can result in large errors as previously described. Convection may be reduced by carrying out the reaction in a constant temperature bath. However, this procedure presents additional equipment requirements for the clinical laboratory. For measurement of hapten antigens, the system is additionally suboptimal if the reaction is carried out at 37.degree. C. according to the manufacturer's recommendation. It has been shown that increasing the temperature of certain antibody-hapten reactions tends to enhance the rate of dissociation of the antibody-hapten complex relative to the rate of its formation. See Smith, T. W. and Skubitz, K. M., Biochemistry 14, 1946, (1975) and Keane, P. M., Walker, W. H. C., Gauldie, J. and Abraham, G. E., Clin. Chem. 22, 70 (1976).
One system which affords a high surface area for over-all volume is the coated micro glass bead system as, for example, the Immo Phase.TM. system of Corning Glass Works. This system exemplifies the use of finely divided particles. It provides a high coated surface area with a correspondingly high reaction rate. Due to settling of the particles during the reaction, optimization of test systems of this kind requires that the test tubes in which they are placed during reaction be capped and mixed vertically during the reaction to insure that all surfaces come in contact with the reactants. Further, the use of particles necessitates multiple centrifugations and washings to completely separate the immobilized product from the solution reactants. Glass particle surfaces have the further disadvantage that there is greater nonspecific protein binding to glass, as compared to plastic.
Coated tubes, wherein the reagents are coated covalently or by adsorption on the inner surfaces thereof, (as described, for example, in U.S. Pat. No. 3,867,517, issued Feb. 18, 1975 to Ling) are easy to use. They offer a more limited surface area than the present invention and their correspondingly slow reaction time may decrease the specificity of the prescribed test procedure.
A third type of solid phase insert matrix is represented by the StiQ.TM. assay of International Diagnostic Technology Corporation, Santa Clara, Calif., designed to exploit a solid phase assay disclosed in U.S. Pat. No. 4,020,151, issued Apr. 26, 1977 to Bolz, et al. In this system, a disc shaped, uncoated insert matrix of material capable of adsorbing proteins from serum is provided. In this system, the limitations are not only due to surface to volume ratio or geometric considerations but are mainly due to problems associated with the initial adsorption step: the presence of interfering substances and the difficulty of obtaining measurable adsorption of components present in low concentration.
Sponge-like matrices, while offering relatively large surface areas, are difficult to wash thoroughly at the conclusion of the reaction time, thus making separation extremely difficult. See U.S. Pat. No. 3,951,748 issued Apr. 20, 1976 to Devlin. U.S. Pat. No. 3,464,798 issued Sept. 2, 1969 to Kilthau discloses a combination of a receptacle and a closely-fitting insert matrix, so shaped as to squeeze the reaction fluid into a thin layer between the container walls and the matrix surface.
Another possibly pertinent patent, though not employing a solid phase matrix is U.S. Pat. No. 3,206,602 issued Sept. 14, 1965 to Eberle.